Metal affinity partitioning (Johansson, G. 1985. Partitioning of proteins, pp. 161-219. In: H. Walter, D. E. Brooks, and D. Fisher (eds.), Partitioning in aqueous two-phase systems. Academic Press, Orlando) exploits the affinity of transition metal ions for electron-rich amino acid residues, such as histidine and cysteine, accessible on the surfaces of some proteins. When the metal ion is partially chelated and coupled to a linear polymer, such as polyethylene glycol ("PEG"), the resulting polymer-bound metal chelate can be used to enhance the partitioning of metal binding proteins into the polymer-rich phase of a PEG-salt or PEG-dextran aqueous two-phase system. Since most proteins favor the salt-rich heavy phase of an aqueous two-phase system, metal affinity partitioning may be a very efficient and selective means of isolating and purifying a metal-binding protein from a crude mixture, although, prior to the instant invention, this was not done.
The application of a metal affinity ligand for the isolation of proteins is set forth in Porath, et al. (Porath, J., Carlsson, J., Olsson, L., Belfrage, G. 1975. Nature 258:598-599) who demonstrated that histidine- and cysteine-containing proteins could be chromatographically separated from each other using a support that had been functionalized with a chelator, such as iminodiacetic acid ("IDA"), which is attached to a polymer spacer and bound to a metal such as copper, zinc or nickel. Immobilized metal affinity chromatography ("IMAC") has evolved into a very powerful tool for protein chromatography and a number of IDA-based IMAC resins are now commercially available (Sulkowski, E. 1985. Purification of proteins by IMAC. Trends Biotechnol. 3:1-7).
Metal affinity partitioning of previously purified proteins has been reported by Wuenschell, et al. (Wuenschell, G. E., Naranjo, E., Arnold, F. H. 1990. Bioprocess Eng. 5:199-202) who studied the partitioning of selected purified heme-containing proteins in an aqueous two-phase system of PEG and dextran using Cu(II)IDA-PEG as the affinity ligand. They showed that protein partitioning was affected by the number of accessible histidine residues on the protein surface.
Birkenmeier, et al. (Birkenmeier, G., Vijayalakshmi, M. A., Stigbrand, T., Kopperschlager, G. 1991. J. Chrom. 539:267-277) investigated the relative affinities of several transition metal ions by comparing the partitioning of purified .alpha..sub.2 -macroglobulin, tissue plasminogen activator, superoxide dismutase, and monoclonal antibodies using IDA-PEG chelated with Cu(II), Ni(II), Zn(II) and Fe(III) in an aqueous two-phase system of PEG and dextran. Their findings suggested that copper is the most effective transition metal ion for metal affinity partitioning. However, the use of copper may be problematical. Cu(II)-catalyzed oxidation of heme-proteins has been well-characterized by Rifkind (Rifkind, J. M., 1974. Biochemistry, 13:2475-2481) who showed that, in the absence of a strong chelator such as EDTA, autoxidation of heme proteins occurs rapidly, within 1-3 minutes, with as little as 10.sup.-3 molar equivalents of Cu(II). Hemoglobin undergoes rapid oxidation by Cu(II) but is unaffected by Ni(II), Zn(II) and other common transition metal ions. Heme oxidation is initiated by the binding of Cu(II) to a high affinity copper binding site followed by the dissociation of the sixth liganded position of the heme (Rifkind, JM 1976. Biochemistry 15: 5337-5343). Oxidation of the heme iron from Fe(II) to Fe(III) can result in an irreversible loss of oxygen binding ability. Furthermore, unchelated Cu(II) in a protein solution can cause protein precipitation (Nagel, R. L., Bemski, G., Pincus, P., 1970. Arch. Biochem. Biophys. 137: 428-434). Metal catalyzed oxidation ("MCO") reactions during immobilized metal affinity chromatography with Cu(II)IDA-linked resins have been recently reported (Krishnamurthy, R., Madurawe, R. D., Bush, K. D., Lumpkin, J. A. 1995. Biotechnol. Prog. 11:643-650).
Protein partitioning has been shown by Suh and Arnold (Suh, S.-S., Arnold, F. H. 1990. Biotechnol. Bioeng. 35:682-690) to depend on ligand concentration, pH, number and pK.sub.a of accessible surface histidines, and the association constant for the binding of the metal ligand to the unprotonated histidine side chains. They used these observations to develop a comprehensive model for metal affinity partitioning. Arnold and her coworkers have partitioned other purified proteins using IDA-PEG including recrystallized human hemoglobin with Cu(II)IDA-PEG as the metal ligand (Plunkett, S. D., Arnold, F. H. 1990. Biotechnol. Tech. 4:45-48) and phosphoproteins, such as egg yolk phosvitin, with Fe(III) IDA-PEG as the metal ligand (Chung, B. H., Arnold, F. H. 1991. Biotechnol. Let. 13:615-620).
Enhanced partitioning of a protein also depends on the spatial arrangement of electron-rich amino acid residues on the protein surface. For example, two histidines separated by three amino acids on an .alpha.-helix exhibit a particularly high metal binding affinity (Sulkowski, E. 1987. pp. 149-162. In Protein purification: micro to macro, R. Burgess (ed.), A. R. Liss, Inc. New York). Metal binding sites containing the His-X.sub.3 -His motif have been engineered onto the surface of iso-1-cytochrome c (Todd, R. J., Van Dam, M. E., Casimiro, D., Haymore, B. L., Arnold, F. H. 1991. Proteins: Structure, Function, and Genetics 10:156-161) and into hirudin (Chung, B. H., Sohn, J. H., Rhee, S.-K., Chang, Y. K., Park, Y. H. 1994. J. Ferm. Bioeng. 77:75-79) a 65-amino acid peptide, to enhance partitioning with Cu(II)IDA-PEG in aqueous two-phase systems. All of the studies listed above involved single-protein partitioning using protein that was previously isolated and purified by conventional methods.
While the ability of metal chelates to recognize and partition metal-binding proteins has been well-documented in artificial settings such as those described above, development of metal affinity partitioning into a useful technology for the isolation and purification of proteins from crude solutions was not done prior to the present invention. There are many problems to overcome in using metal chelates to purify a target protein from a crude preparation. In particular with heme-containing proteins, there is the oxidation problem referred to above and the question of how selective the ligand is for the target protein. There also is a problem of nitrogen-containing compounds in the crude system inhibiting ligand binding to the target protein. Finally, there is a problem relating to protein solubility and potential precipitation of proteins by the salt used in an aqueous two-phase partitioning system. Applicant has overcome these and other problems to develop a functional aqueous two-phase metal affinity partitioning system for purifying target proteins from crude protein solutions.